U.S. patent application number 14/793566 was filed with the patent office on 2015-10-29 for high sensitivity electrospray interface.
This patent application is currently assigned to University of Notre Dame de Lac. The applicant listed for this patent is University of Notre Dame de Lac. Invention is credited to Norman Dovichi, Liangliang Sun, Guijie Zhu.
Application Number | 20150311056 14/793566 |
Document ID | / |
Family ID | 52587387 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150311056 |
Kind Code |
A1 |
Dovichi; Norman ; et
al. |
October 29, 2015 |
HIGH SENSITIVITY ELECTROSPRAY INTERFACE
Abstract
The invention provides a sheath-flow interface for producing
electrospray from a capillary. The electrospray generated by the
interface can be used as the source of ions for mass spectrometry.
Electrokinetic flow in the interface can move a sheath liquid past
the end of a capillary so as to mix with an analyte effluent
discharged from the capillary. The sheath liquid and analyte
mixture can be directed to an electrospray emitter to generate an
electrospray.
Inventors: |
Dovichi; Norman; (South
Bend, IN) ; Sun; Liangliang; (South Bend, IN)
; Zhu; Guijie; (South Bend, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Notre Dame de Lac |
Notre Dame |
IN |
US |
|
|
Assignee: |
University of Notre Dame de
Lac
Notre Dame
IN
|
Family ID: |
52587387 |
Appl. No.: |
14/793566 |
Filed: |
July 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2014/053522 |
Aug 29, 2014 |
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14793566 |
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61871562 |
Aug 29, 2013 |
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Current U.S.
Class: |
250/282 ;
250/288; 250/423R; 250/424 |
Current CPC
Class: |
G01N 30/7266 20130101;
H01J 49/004 20130101; H01J 49/0445 20130101; H01J 49/0404 20130101;
H01J 49/167 20130101; G01N 27/44743 20130101; F04B 19/006 20130101;
G01N 27/4473 20130101; G01N 27/4473 20130101; G01N 30/7266
20130101 |
International
Class: |
H01J 49/16 20060101
H01J049/16; H01J 49/00 20060101 H01J049/00; G01N 30/72 20060101
G01N030/72; H01J 49/04 20060101 H01J049/04; G01N 27/447 20060101
G01N027/447 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. R01GM096767 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A sheath-flow interface for producing electrospray from a
capillary comprising: (a) a capillary configured to contain an
analyte liquid, the capillary having an injection end configured to
receive the analyte liquid and a distal end configured to expel
analyte effluent, wherein the outer diameter of a segment of the
distal end tapers to a reduced outer diameter within the range of
about 20 .mu.m to about 200 .mu.m; (b) an electrospray emitter
coaxially disposed surrounding at least the distal end of the
capillary, the electrospray emitter having a distal end that is
tapered to terminate at an opening, the opening being coaxially
disposed in relation to the distal end of the capillary; and (c) a
sheath liquid reservoir in liquid communication with an interior of
the electrospray emitter, such that an electrically conductive
sheath liquid can flow from the sheath liquid reservoir, through a
connecting fixture intermediate the capillary and the electrospray
emitter, across the distal end of the capillary, and through the
opening at the distal end of the electrospray emitter; wherein the
sheath liquid provides electrical contact between the capillary and
the electrospray emitter, the sheath-flow interface is configured
to produce a nanospray generated by electrokinetic flow of the
sheath liquid mixed with the analyte effluent, and the
electrokinetic flow is generated by an electric potential between
the electrospray emitter and a target surface disposed adjacent,
but not in physical contact with, the opening of the emitter.
2. The sheath-flow interface of claim 1 wherein the distal end of
the capillary is about 750 .mu.m from the distal end of the emitter
orifice to about 100 .mu.m beyond the emitter orifice opening.
3. The sheath-flow interface of claim 2 wherein the distal end of
the capillary is about 100 nm to about 750 .mu.m from the distal
end of the emitter orifice.
4. The sheath-flow interface of claim 2 wherein the distal end of
the capillary extends to within the termination point of the distal
end of the emitter orifice to about 100 .mu.m beyond the distal end
of the emitter orifice.
5. The sheath-flow interface of claim 1 wherein the outer diameter
of the distal end of the capillary is about 20 .mu.m to about 75
.mu.m.
6. The sheath-flow interface of claim 1 wherein the segment of the
distal end that tapers to a reduced outer diameter comprises a
segment length of about 0.1 mm to about 10 mm.
7. The sheath-flow interface of claim 1 wherein the sheath-flow
interface can detect a plurality of peptides from a 400 femtogram
sample of peptides in less than 12 minutes when configured with
capillary zone electrophoresis and a tandem mass spectrometer.
8. The sheath-flow interface of claim 7 wherein the peptides
comprise an E. coli tryptic digest.
9. The sheath-flow interface of claim 7 wherein the mass
spectrometer detection limit is about 1-10 zeptomoles.
10. The sheath-flow interface of claim 7 wherein the mass
spectrometer detection limit is approximately 1 zeptomole.
11. The sheath-flow interface of claim 1 wherein the sheath-flow
interface is configured with a capillary zone electrophoresis
instrument and a mass spectrometer, wherein greater than 150
peptides can be identified by accurate mass and time tags from
sub-picogram amounts of a complex protein digest in less than 12
minutes of mass spectrometer time.
12. A method to analyze a protein digest comprising configuring the
sheath-flow interface of claim 1 with a capillary zone
electrophoresis instrument, wherein the analyte liquid is separated
within a separation capillary by capillary zone electrophoresis,
and about 10 kV of potential is applied to provide an electric
field of about 300 V/cm, to produce a wide analyte separation
window, during which time analytes migrate from the capillary into
the interface within about 60 minutes.
13. The method of claim 12 wherein an average of 250,000
theoretical plates to about 350,000 theoretical plates are obtained
for peptide separations.
14. The method of claim 12 wherein the inner diameter of the
separation capillary of the sheath-flow interface is about 5 .mu.m
to about 75 .mu.m.
15. The method of claim 12 wherein the total flow rate for spray is
about 20-200 nL/minute.
16. The method of claim 12 comprising configuring the sheath-flow
interface of claim 1 with tandem mass spectrometry, wherein the
sheath-flow interface is configured to provide the nanospray to a
mass spectrometer for analysis, wherein the target surface is an
input orifice of the mass spectrometer, and wherein the lower
detection limit of protein samples is about 3 femtograms about 5
femtograms.
17. The method of claim 16 wherein the mass detection limit of
peptides analyzed is about 1 zeptomole.
18. The method of claim 16 wherein the signal-to-noise ratio of
peptides analyzed is about 260:1 to about 300:1.
19. A method for producing a nanospray of an analyte effluent from
a capillary using a sheath-flow interface according to claim 1,
comprising applying a voltage to the sheath liquid reservoir
sufficient to drive electroosmotic flow of the sheath liquid from
the sheath liquid reservoir, through a connecting fixture
intermediate the capillary and the electrospray emitter, across the
distal end of the capillary, and through the opening at the distal
end of the electrospray emitter, wherein the analyte effluent is
separated within the capillary by capillary zone electrophoresis by
applying a voltage to the injection end of the capillary.
Description
RELATED APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. 111(a) of
International Application No. PCT/US2014/053522 filed Aug. 29, 2014
and published in English as WO 2015/031820 on Mar. 5, 2015, which
claims priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application No. 61/871,562, filed Aug. 29, 2013, which applications
and publication are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Bottom-up proteomics is a useful method to identify proteins
and characterize their amino acid sequences and post-translational
modifications by proteolytic digestion of proteins prior to
analysis by mass spectrometry. Bottom-up proteomics is widely used
for qualitative and quantitative characterization of complex
biological samples. Given micrograms of material, it is possible to
identify more than 10,000 proteins from mammalian cell lysates and
over 2,500 proteins from prokaryote lysates. The performance of
bottom-up proteomics degrades rapidly for mass-limited samples,
such as laser capture microdissected tissues, circulating tumor
cells, single embryos, and single somatic cells.
[0004] There have been a handful of reports of bottom-up proteomics
of nanogram samples using capillary liquid chromatography
(LC)-electrospray ionization (ESI)-tandem mass spectrometry
(MS/MS). Mann's group identified 2,000 proteins from single
pancreatic islets with protein content of several hundred ng
(Waanders et al., Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18902).
Karger's group identified 566 proteins from 50 ng of digest of
Methanosarcina acetivorans (Yue et al., Anal. Chem. 2007, 79, 938)
and 163 proteins from .about.2.5 ng of the tryptic digest of a
cervical cancer cell line (Luo et al., Anal. Chem. 2007, 79, 6174).
Smith's group detected 870 proteins with an accurate mass and time
tags (AMTs).sup.[8] strategy from low nanogram amounts of the
digest of Deinococcus radiodurans (Smith et al., Proteomics 2002,
2, 513). Smith's group also reported the detection of the three
most abundant proteins in a 0.5 pg sample with the AMTs method, and
reported a .about.10 zmole detection limit for one peptide in a
bovine serum albumin digest (Shen et al., Anal. Chem. 2004, 76,
144). Dovichi and co-workers used a Q-Exactive mass spectrometer
with higher energy collisional dissociation (Olsen et al., Nat.
Methods 2007, 4, 709) to identify .about.100 protein groups from 1
ng of a digest of the RAW264.7 macrophage cell line (Sun et al.,
Rapid Commun. Mass Spectrom. 2013, 27, 157). All of these analyses
required at least one hour of instrument time. Thus, faster methods
for bottom-up analysis of femtogram amounts of protein digest would
be a significant benefit to the proteomics community.
[0005] Capillary zone electrophoresis-electrospray
ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has attracted
attention for bottom-up proteomics, and this approach consistently
outperforms LC-MS/MS for low nanogram samples. The improved
performance of CZE for small sample amounts presumably is due to
its very simple design, eliminating sample loss on injectors and
fittings. Beginning with the pioneering work of Smith's group
(Smith et al., Anal. Chem. 1988, 60, 1948), electrospray interfaces
have been developed for capillary electrophoresis (Maxwell et al.,
Anal. Chim. Acta 2008, 627, 25). However, new interfaces are needed
to reduce sample dilution due to low sheath flow rates, to
eliminate mechanical pumps, to tolerate a wide range of separation
buffers, and to stabilize operation in the nanospray regime.
SUMMARY
[0006] The invention provides an ultrasensitive and fast capillary
zone electrophoresis-electrospray ionization-tandem mass
spectrometry (CZE-ESI-MS/MS) system based on an improved
electrokinetically-pumped sheath-flow interface. The interface has
several advantages, including reduced sample dilution due to a very
low sheath flow rate, elimination of mechanical pumps, tolerance
for a wide range of separation buffers, compatibility with
commercial capillary electrophoresis instruments, and stable
operation in the nanospray regime. The system is useful for rapid
bottom-up analysis of femtogram amounts of protein digests and
provides extremely high sensitivity.
[0007] Accordingly, the invention provides a sheath-flow interface
for producing electrospray from a capillary, wherein the interface
includes:
[0008] (a) a capillary configured to contain an analyte liquid, the
capillary having an injection end configured to receive the analyte
liquid and a distal end configured to expel analyte effluent
wherein the outer diameter of a segment of the distal end tapers to
a reduced outer diameter in the range of about 20 .mu.m to about
200 .mu.m;
[0009] (b) an electrospray emitter coaxially disposed surrounding
at least the distal end of the capillary, the electrospray emitter
having a distal end that is tapered to terminate at an opening, the
opening being coaxially disposed in relation to the distal end of
the capillary; and
[0010] (c) a sheath liquid reservoir in liquid communication with
an interior of the electrospray emitter, such that an electrically
conductive sheath liquid can flow from the sheath liquid reservoir,
through a connecting fixture intermediate the capillary and the
electrospray emitter, across the distal end of the capillary, and
through the opening at the distal end of the electrospray
emitter.
[0011] The sheath-flow interface can be configured such that the
distal end of the capillary and the electrospray emitter orifice
are separated by a distance of about 750 .mu.m to about 100 nm
within the electrospray emitter. In embodiments where the outer
diameter of the capillary is smaller than the emitter orifice
diameter, the distal end of the capillary can be configured to
reside within the emitter orifice opening. Furthermore, the distal
end of the capillary can extend up to about 100 .mu.m beyond the
emitter orifice opening.
[0012] The sheath liquid can provide electrical contact between the
capillary and the electrospray emitter. The sheath-flow interface
can be configured to produce a nanospray generated by
electrokinetic flow of the sheath liquid mixed with the analyte
effluent. Furthermore, the electrokinetic flow can be generated by
an electric potential between the electrospray emitter and a target
surface disposed adjacent, but not in physical contact with, the
opening of the emitter.
[0013] In various embodiments, the outer diameter of the distal end
of the capillary can be about 20 .mu.m to about 75 .mu.m, or about
45 .mu.m to about 65 .mu.m. The segment of the distal end that
tapers to a reduced outer diameter can be a segment length of about
0.1 mm to about 10 mm, typically about 5 mm.
[0014] The sheath-flow interface can detect a plurality of peptides
from a 400 femtogram sample of peptides in less than 12 minutes, or
less than 10 minutes, of mass spectrometry instrument time when
configured with capillary zone electrophoresis and a tandem mass
spectrometer. The peptides comprise peptide digests such as an E.
coli tryptic digest.
[0015] The sheath-flow interface can be configured with a capillary
zone electrophoresis instrument and a mass spectrometer, wherein
greater than 150 peptides can be identified by accurate mass and
time tags from sub-picogram amounts of a complex protein digest in
less than 12 minutes of mass spectrometer time, often in less than
10 minutes of mass spectrometer time.
[0016] The mass spectrometer detection limit can be about 1-10
zeptomoles, for example, as low as approximately 1 zeptomole.
[0017] The invention also provides methods to analyze a protein
digest comprising configuring the sheath-flow interface of claim 1
with a capillary zone electrophoresis instrument, wherein the
analyte liquid is separated within a separation capillary by
capillary zone electrophoresis, and about 10 kV of potential is
applied to provide an electric field of about 300 V/cm, to produce
a wide analyte separation window, during which time analytes
migrate from the capillary into the interface within about 60
minutes. An average of 250,000 theoretical plates to about 350,000
theoretical plates are obtained for peptide separations.
[0018] In some embodiments, the inner diameter of the separation
capillary of the sheath-flow interface is about 5 .mu.m to about 75
.mu.m. In various embodiments, the total flow rate for spray is
about 15 to about 200 nL/minute, about 20 to about 200 nL/minute,
about 15 to about 25 nL/minute, or about 20 nL/minute. The methods
can include configuring the sheath-flow interface described herein
with tandem mass spectrometry, wherein the sheath-flow interface is
configured to provide the nanospray to a mass spectrometer for
analysis, wherein the target surface is an input orifice of the
mass spectrometer, and wherein the lower detection limit of protein
samples is about 3 femtograms about 5 femtograms. The mass
detection limit of peptides analyzed can be about 1 zeptomole,
particularly when a small inner diameter separation capillary
(e.g., less than about 15 microns, typically about 10 microns, in
diameter). To achieve the very low mass detection limit, a highly
sensitive mass spectrometer should be employed. One example of a
highly sensitive mass spectrometer is the Q-Exactive
Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific
Inc.). The signal-to-noise ratio of peptides analyzed can be about
260:1 to about 300:1.
[0019] The invention further provides methods for producing a
nanospray of an analyte effluent from a capillary using a
sheath-flow interface described herein comprising applying a
voltage to the sheath liquid reservoir sufficient to drive
electroosmotic flow of the sheath liquid from the sheath liquid
reservoir, through a connecting fixture intermediate the capillary
and the electrospray emitter, across the distal end of the
capillary, and through the opening at the distal end of the
electrospray emitter, wherein the analyte effluent is separated
within the capillary by capillary zone electrophoresis by applying
a voltage to the injection end of the capillary.
[0020] In various embodiments, the analyte liquid can be moved
through the capillary by an electrokinetic force or a mechanical
pumping force. The analyte liquid can be separated within the
capillary by capillary zone electrophoresis, micellar
electrokinetic chromatography, capillary electrochromatography,
capillary isoelectrofocusing, capillary liquid chromatography, or
combinations thereof. The nanospray can be produced by, for
example, electroosmotic flow. The analyte liquid can be separated
within the capillary by electrophoresis, wherein the nanospray is
produced by electroosmotic flow, and wherein both the
electrophoresis and the electroosmotic flow are driven by applying
an electric potential between the injection end of the capillary,
the sheath liquid reservoir, and the target surface. The analyte
liquid can be separated by chromatography where flow is driven by
either a mechanical pump in conventional liquid chromatography or
by electrokinetic flow in electrochromatograpy; in these cases,
electrospray can be driven by electroosmotic flow within the
emitter.
[0021] The sheath-flow interface can be configured to provide the
nanospray to a mass spectrometer for analysis, and wherein the
target surface is an input orifice of the mass spectrometer. The
target surface can be held at ground, or the target surface can be
held at a potential. The sheath liquid can be configured to enhance
the compatibility of the analyte effluent with the mass
spectrometer. The opening in the distal end of the electrospray
emitter can be about 0.5 .mu.m to about 50 .mu.m in diameter, or
about 0.5 .mu.m to about 30 .mu.m in diameter.
[0022] The invention also provides methods for analyzing
biomolecules such as a protein digest. The methods can include
forming an analyte effluent from a protein digest and producing a
nanospray of the analyte effluent from a capillary using an
embodiment of the sheath-flow interface described above. The
methods of producing the nanospray of the analyte effluent can
include applying a voltage to the sheath liquid reservoir
sufficient to drive electroosmotic flow of the sheath liquid from
the sheath liquid reservoir, through a connecting fixture
intermediate the capillary and the electrospray emitter, across the
distal end of the capillary, and through the opening at the distal
end of the electrospray emitter. The analyte effluent can be
separated within the capillary by capillary zone electrophoresis by
applying a voltage to the injection end of the capillary.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the specification and
are included to further demonstrate certain embodiments or various
aspects of the invention. In some instances, embodiments of the
invention can be best understood by referring to the accompanying
drawings in combination with the detailed description presented
herein. The description and accompanying drawings may highlight a
certain specific example, or a certain aspect of the invention.
However, one skilled in the art will understand that portions of
the example or aspect may be used in combination with other
examples or aspects of the invention.
[0024] FIGS. 1A and 1B. (FIG. 1A) Schematic illustration of a
representative interface in accordance with disclosed embodiments.
(FIG. 1B) Schematic of a representative interface with a mass
spectrometer. The sheath liquid can be pumped by electrokinetic
flow driven by the potential difference between HV2 and an inlet of
the mass spectrometer. The separation can be driven by the
potential difference between the inlet (HV1) and outlet (HV2) of
the capillary.
[0025] FIG. 2. Components of an interface, according to one
embodiment.
[0026] FIG. 3A-C. CZE-ESI-MS/MS system. Sketch of the system (FIG.
3A), sketch of the etched capillary in the electrospray emitter
(FIG. 3B), and micrograph of the etched capillary in the emitter
(FIG. 3C).
[0027] FIG. 4. Extracted ion electropherograms of 50 high intensity
peptides identified based on tandem spectra from 16 pg amounts of
E. coli digests analyzed by CZE-ESI-MS/MS in triplicate. The mass
tolerance for extraction was 2 ppm.
[0028] FIG. 5. Relative standard deviation (RSD) distribution of
peptide migration time for triplicate runs of 16 pg of E. coli
digests. Selected ion electropherograms were generate from 154 high
intensity peptides. The mass tolerance used for peptide peak
extraction was 3 ppm. A Gaussian function was fit to the
electropherograms using an unsupervised nonlinear least squares
fit:
Intensity(t)=a.times.exp-0.5*(t-t.sub.0).sup.2/width.sup.2+offset;
where t is time, t.sub.0 is the peak migration time, a is peak
amplitude, width is the peak's width in terms of the standard
deviation of the Gaussian, and offset is a dc offset. MATLAB's fit
routine was used for the calculation. The amplitude and migration
time for the maximum point in each electropherogram was used for
the initial guesses of a and t.sub.0. The initial guess for width
was 3 seconds. The initial estimate for offset was the median value
of the electropherogram. The relative standard deviation in
migration time was calculated as
std(t.sub.0)/mean(t.sub.0).times.100% for each set of triplicate
electropherograms.
[0029] FIG. 6. Peak width distribution of identified peptides from
16 pg amounts of E. coli digests with CZE-ESI-MS/MS in triplicate.
Peak width was determined from the nonlinear regression analysis
described in the description for FIG. 5 above. The full width at
half height for a Gaussian function=2.35.times.width, and the full
width at baseline is 4.times.width.
[0030] FIG. 7. Distribution of the number of theoretical plates (N)
for peptides identified from 16 pg amounts of E. coli digests with
CZE-ESI-MS/MS in triplicate. The number of theoretical plates was
calculated as N=(t.sub.0/width).sup.2 where t.sub.0 and width were
determined in the nonlinear regression analysis described in the
description for FIG. 5 above.
[0031] FIG. 8A-C. Correlation of peptide intensities between runs
from 16 pg amounts of E. coli digests analyzed (FIG. 8A: peptide
intensity from 16 pg amounts of E. coli.sub.--1.sup.st run vs.
peptide intensity from 16 pg amounts of E. coli.sub.--2.sup.nd run;
FIG. 8B: peptide intensity from 16 pg amounts of E.
coli.sub.--1.sup.st run vs. peptide intensity from 16 pg amounts of
E. coli.sub.--3.sup.rd run; FIG. 8C: peptide intensity from 16 pg
amounts of E. coli.sub.--2.sup.nd run vs. peptide intensity from 16
pg amounts of E. coli.sub.--3.sup.rd run). The peptide intensity
was obtained from database searching results with MaxQuant software
(v. 1.3.0.5), which is the summed eXtracted Ion Current (XIC) of
all isotopic clusters associated with the identified peptide
sequence.
[0032] FIG. 9A-B. Relationship between loaded amounts of E. coli
digests and identifications based on tandem spectra (points
connected by lines) and accurate mass and time tags (star=AMTs).
Protein identifications (FIG. 9A); peptide identifications (FIG.
9B). Each sample was analyzed in duplicate or triplicate. The
identifications based on AMTs from 400 fg amounts of E. coli
digests were labelled with star. The error bars are standard
deviations of the mean.
[0033] FIG. 10. Extracted ion electropherogram of an identified
peptide from 400 fg (top), and the corresponding electropherogram
from a 16 pg amount of E. coli digests (bottom), based on accurate
mass and time tags (AMTs) method (153 additional pair of
electropherograms not shown). The mass tolerance used for peak
extraction was 3 ppm.
[0034] FIG. 11A-C. The extracted ion electropherograms of three
elongation factor Tu peptides (FIGS. 11A, 11B, and 11C) identified
with MS/MS from 400 fg amounts of E. coli digests for calculation
of peptide detection limits.
[0035] FIG. 12. Wide dynamic range calibration curve for loading
amounts and fragment ions (y2.sup.+ and b6.sup.+) intensity of
angiotensin II after CZE-PRM analysis. The lines are the results of
unweighted linear fit to the log-log data.
[0036] FIG. 13. Relative standard deviations (RSDs) of migration
time and intensity of angiotensin II fragment ion (y2.sup.+) for
different loading amounts (2 amole-150 fmole) from triplicate
CZE-PRM analysis.
[0037] FIG. 14A-B. Base peak electropherogram of MCF7 digest after
CZE-ESI-MS analysis. (FIG. 14A) Entire separation; (FIG. 14B)
detail of the separation from 12 to 18 minutes. Data were treated
with a Lowess filter with Gaussian kernel and span of 10 points.
The sample loading amount was .about.60 ng; the capillary was 20
.mu.m i.d. and 100 cm long; the separation buffer was 0.5% (v/v)
FA; and a field strength of 280 V/cm was used for the
separation.
DETAILED DESCRIPTION
[0038] Described herein is an electrokinetically pumped sheath-flow
electrospray interface and the demonstration of its use when
coupled with capillary electrophoresis. For useful capillary
electrophoresis systems, see Wojcik et al., Rapid Commun. Mass
Spectrom. 2010; 24: 2554-60. A significant modification to previous
interfaces is described herein, which modification provides roughly
two orders of magnitude improved sensitivity. The improved
interface has been used for the analysis of a variety of digests,
including 400 femtograms of a tryptic digest of an E. coli lysate.
In that digest, 154 peptides from over 60 proteins were identified
in a 10 minute analysis. Mass detection limits for three peptides
from the same protein are approximately 1 zeptomole (600
molecules). The improved interface produces a two-order of
magnitude improvement in the state of art for bottom-up protein
identification and a one-order of magnitude improvement in the
state of art for MS-based peptide detection limits.
[0039] The importance of the tapered capillary arises in part from
the dimensions of capillaries used in commercial electrophoresis
instruments. Those instruments (e.g., Beckman instruments)
typically use 375 micrometer outer diameter fused silica
capillaries. Those capillaries are far too large to be used with
the improved interface described herein because the standard
capillary tips butt against the emitter when attempting to reduce
the distance from the capillary tip to the emitter orifice. By
modifying (e.g., reducing the outer diameter of) the distal end of
capillaries, they become compatible with the interface described
herein.
Definitions
[0040] The following definitions are included to provide a clear
and consistent understanding of the specification and claims. As
used herein, the recited terms have the following meanings All
other terms and phrases used in this specification have their
ordinary meanings as one of skill in the art would understand. Such
ordinary meanings may be obtained by reference to technical
dictionaries, such as Hawley's Condensed Chemical Dictionary
14.sup.th Edition, by R. J. Lewis, John Wiley & Sons, New York,
N.Y., 2001.
[0041] References in the specification to "one embodiment", "an
embodiment", etc., indicate that the embodiment described may
include a particular aspect, feature, structure, moiety, or
characteristic, but not every embodiment necessarily includes that
aspect, feature, structure, moiety, or characteristic. Moreover,
such phrases may, but do not necessarily, refer to the same
embodiment referred to in other portions of the specification.
Further, when a particular aspect, feature, structure, moiety, or
characteristic is described in connection with an embodiment, it is
within the knowledge of one skilled in the art to affect or connect
such aspect, feature, structure, moiety, or characteristic with
other embodiments, whether or not explicitly described.
[0042] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a compound" includes a plurality of such
compounds, so that a compound X includes a plurality of compounds
X. It is further noted that the claims may be drafted to exclude
any optional element. As such, this statement is intended to serve
as antecedent basis for the use of exclusive terminology, such as
"solely," "only," and the like, in connection with any element
described herein, and/or the recitation of claim elements or use of
"negative" limitations.
[0043] The term "and/or" means any one of the items, any
combination of the items, or all of the items with which this term
is associated. The phrases "one or more" and "at least one" are
readily understood by one of skill in the art, particularly when
read in context of its usage. For example, the phrase can mean one,
two, three, four, five, six, ten, 100, or any upper limit
approximately 10, 100, or 1000 times higher than a recited lower
limit. For example, one or more analytes can refer to one, one or
two, one to three, one to four, one to five, 1-10, 1-100, or 1-500
different analytes or different types of analytes.
[0044] The term "about" can refer to a variation of .+-.5%,
.+-.10%, .+-.20%, or .+-.25% of the value specified. For example,
"about 50" percent can in some embodiments carry a variation from
45 to 55 percent. For integer ranges, the term "about" can include
one or two integers greater than and/or less than a recited integer
at each end of the range. Unless indicated otherwise herein, the
term "about" is intended to include values, e.g., weight
percentages, proximate to the recited range that are equivalent in
terms of the functionality of the individual ingredient, the
composition, or the embodiment. The term about can also modify the
end-points of a recited range as discuss above in this
paragraph.
[0045] As will be understood by the skilled artisan, all numbers,
including those expressing quantities of ingredients, properties
such as molecular weight, reaction conditions, and so forth, are
approximations and are understood as being optionally modified in
all instances by the term "about." These values can vary depending
upon the desired properties sought to be obtained by those skilled
in the art utilizing the teachings of the descriptions herein. It
is also understood that such values inherently contain variability
necessarily resulting from the standard deviations found in their
respective testing measurements.
[0046] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges recited herein also encompass any and all
possible sub-ranges and combinations of sub-ranges thereof, as well
as the individual values making up the range, particularly integer
values. A recited range (e.g., weight percentages or carbon groups)
includes each specific value, integer, decimal, or identity within
the range. Any listed range can be easily recognized as
sufficiently describing and enabling the same range being broken
down into at least equal halves, thirds, quarters, fifths, or
tenths. As a non-limiting example, each range discussed herein can
be readily broken down into a lower third, middle third and upper
third, etc. As will also be understood by one skilled in the art,
all language such as "up to", "at least", "greater than", "less
than", "more than", "or more", and the like, include the number
recited and such terms refer to ranges that can be subsequently
broken down into sub-ranges as discussed above. In the same manner,
all ratios recited herein also include all sub-ratios falling
within the broader ratio. Accordingly, specific values recited for
radicals, substituents, and ranges, are for illustration only; they
do not exclude other defined values or other values within defined
ranges for radicals and substituents.
[0047] One skilled in the art will also readily recognize that
where members are grouped together in a common manner, such as in a
Markush group, the invention encompasses not only the entire group
listed as a whole, but each member of the group individually and
all possible subgroups of the main group. Additionally, for all
purposes, the invention encompasses not only the main group, but
also the main group absent one or more of the group members. The
invention therefore envisages the explicit exclusion of any one or
more of members of a recited group. Accordingly, provisos may apply
to any of the disclosed categories or embodiments whereby any one
or more of the recited elements, species, or embodiments, may be
excluded from such categories or embodiments, for example, for use
in an explicit negative limitation.
[0048] The term "contacting" refers to the act of touching, making
contact, or of bringing to immediate or close proximity, including
at the cellular or molecular level, for example, to bring about a
physiological reaction, a chemical reaction, or a physical change,
e.g., in a solution, in a reaction mixture, in vitro, or in
vivo.
[0049] An "effective amount" refers to an amount effective to bring
about a recited effect, such as an amount necessary to form
products in a reaction mixture. Determination of an effective
amount is typically within the capacity of persons skilled in the
art, especially in light of the detailed disclosure provided
herein. The term "effective amount" is intended to include an
amount of a compound or reagent described herein, or an amount of a
combination of compounds or reagents described herein, e.g., that
is effective to form products in a reaction mixture. Thus, an
"effective amount" generally means an amount that provides the
desired effect, such as the minimum amount of a protein to obtain a
mass spectrogram.
[0050] The term "nanospray" refers to a method of creating an
aerosol of sub-micrometer-sized droplets, or the product of such
process. Nanospray is a form of electrospray ionization in which an
electrostatic field overcomes the surface tension of a liquid to
form a liquid jet. Nanospray can employ glass capillaries with
micrometer-sized exits and flow rates in the nL/minute range.
[0051] The term "electroosmotic flow" refers to the motion of
liquid induced by an applied potential across a capillary tube,
microchannel, or other fluid conduit.
Sheath-Flow Electrospray Interface
[0052] Hyphenation of capillary electrophoresis (CE) with
electrospray ionization mass spectrometry (MS) was developed in the
late 1980s and the combined use of the techniques has steadily
developed since that time. Capillary electrophoresis-electrospray
ionization interfaces generally fall into three categories:
sheathless, co-axial sheath flow, and liquid junction
interfaces.
[0053] The sheath flow interface uses a coaxial sheath liquid that
mixes with analytes as they migrate from the separation capillary.
The sheath liquid provides electrical contact between the
electrophoretic separation and the electrospray ionization source.
The sheath liquid can also modify the separation buffer to make it
more compatible with MS detection. In the now commercially
available sheath flow interface, the distal end of the separation
capillary is inserted within a concentric tube, with the
capillary's end extending beyond the tube. Electrical contact is
made by a sheath liquid flowing over the capillary protruding from
the tube, and a nebulizer gas is supplied to assist spray
formation. The sheath liquid is pumped at a rate that maintains a
stable spray and the interface operates at relatively high sheath
flow rates, typically in the range of a few to several microliters
per minute, which can result in significant sample dilution.
[0054] In a liquid junction interface, the separation capillary and
electrospray emitter orifice are separated by a small gap.
Electrical contact is made with this gap to drive the electrospray.
Unfortunately, the gap can contribute to a loss of separation
efficiency.
[0055] A sheathless interface eliminates sample dilution associated
with the sheath liquid, which tends to result in higher
sensitivity. In sheathless interfaces, the separation capillary
often serves as the electrospray emitter. Ongoing research in the
design of sheathless interfaces has focused on establishing
electrical contact at the distal end of the separation capillary.
Variations include coating the outer tip of the capillary with
metal, inserting an electrode inside the capillary outlet, using
porous etched capillary walls, and using a microdialysis junction.
The major drawback of a sheathless interface is spray instability
due to the very low flow rates produced from separation conditions,
as well as the limited choices of separation buffers due to lack of
post column chemistry.
[0056] To overcome some of the problems associated with both the
original sheath flow and sheathless interface designs, a low flow
version of a sheath flow interface was introduced. In this design,
the separation capillary was inserted inside a tapered glass
emitter. A second capillary was inserted inside the emitter,
supplying sheath liquid, pumped at the rate of 1 .mu.L/min.
Electrospray voltage was supplied by a stainless steel wire
inserted into the emitter.
[0057] Sheath flow interfaces with tapered emitters can operate in
the nanospray regime, which is associated not only with supporting
lower flow rates but also with better desolvation, enhanced
sensitivity, and increased salt tolerance. However, attempting to
produce a nanospray from CE effluent presents many technical issues
that must be overcome in order to advance the field of capillary
electrophoresis-mass spectrometry (CE-MS). Described herein are
systems and methods to overcome these issues.
[0058] Accordingly, in one embodiment, the invention provides a
sheath-flow interface for producing electrospray from a capillary.
The interface can include (a) a capillary configured to contain an
analyte liquid, the capillary having an injection end configured to
receive the analyte liquid and a distal end configured to expel
analyte effluent, wherein the outer diameter of a segment of the
distal end tapers to a reduced outer diameter in the range of about
20 .mu.m to about 200 .mu.m, about 20 .mu.m to about 100 .mu.m, or
about 40 .mu.m to about 100 .mu.m; (b) an electrospray emitter
coaxially disposed surrounding at least the distal end of the
capillary, the electrospray emitter having a distal end that is
tapered to terminate at an opening, the opening being coaxially
disposed in relation to the distal end of the capillary; and (c) a
sheath liquid reservoir in liquid communication with an interior of
the electrospray emitter, such that an electrically conductive
sheath liquid is allowed to flow from the sheath liquid reservoir,
through a connecting fixture intermediate the capillary and the
electrospray emitter, across the distal end of the capillary, and
through the opening at the distal end of the electrospray
emitter.
[0059] The sheath liquid can provide electrical contact between the
capillary and the electrospray emitter. The sheath-flow interface
can be configured to produce a nanospray generated by
electrokinetic flow of the sheath liquid mixed with the analyte
effluent. The electrokinetic flow can be generated by an electric
potential between the electrospray emitter and a target surface
disposed adjacent, but not in physical contact with, the opening of
the emitter. The interface can thus be used to produce electrospray
from a capillary, which electrospray can be used as a source of
ions for mass spectrometry analysis or other analytical techniques
that can make use of an electrospray such as evaporative light
scatter detection, inductively coupled plasma detection, and
electrospray deposition of target molecules on a surface for
subsequent analysis.
[0060] The sheath-flow interface can be further described with
reference to FIG. 1A. The interface 100 includes a capillary 102.
The capillary 102 includes an injection end 104 that is configured
to receive an analyte liquid. The analyte liquid can be introduced
into the capillary 102 at the injection end 104 by various means
known to those of skill in the art. For example, the injection end
104 can be interfaced with a chromatography column, such that
effluent from the chromatography column is injected into the
capillary 102. Other methods for providing analyte to the capillary
102 include contacting the injection end 104 with a reservoir of
analyte and performing electrokinetic, hydrostatic or hydrodynamic
injection.
[0061] The capillary 102 has a distal end 106 that is configured to
expel analyte effluent. The outer surface diameter of distal end
106 is reduced compared to the outer diameter of the rest of
capillary 102. The distal segment of distal end 106 (e.g., the last
0.1-10 mm of the capillary 102) can have an outer diameter that is
reduced by about 30% to about 90%, or by about 40% to about 75%,
often by about 60%, while the inner diameter remains approximately
the same. Thus, in embodiments where the outer diameter of
capillary 102 is about 150 .mu.m, the outer diameter of distal end
106 can be reduced to an outer diameter of about 20 .mu.m to about
200 .mu.m, about 20 .mu.m to about 100 .mu.m, about 40 .mu.m to
about 100 .mu.m, about 60 .mu.m, or about 50 .mu.m.
[0062] Typically, the outer diameter of the distal end of the
capillary is chemically reduced without affecting the inner
diameter. In other embodiments, the inner diameter can also be
reduced, for example, when the outer diameter is reduced by a
heating and pulling technique. Other techniques for reducing the
outer diameter of the distal end of the capillary include
mechanical grinding and sand blasting.
[0063] Analyte effluent, as used herein, refers to the analyte that
has passed through the length of the capillary 102 and is expelled
from the distal end 106. The analyte effluent can be moved through
the capillary 102 by various means such as by using an
electrokinetic force (e.g., electroosmotic and/or electrophoretic
flow).
[0064] Disposed around the capillary 102 is an electrospray emitter
110. The electrospray emitter is coaxially disposed around at least
the distal end 106 of the capillary 102, such that the capillary
102 and an opening 114 of the electrospray emitter 110 are
coaxially arranged along axis A, as illustrated in FIG. 1A. A
distal end 112 of the electrospray emitter 110 is tapered from the
tubular cylindrical body of the emitter 110 in order to form the
opening 114.
[0065] Electrospray can be generated from the analyte effluent
passing through the capillary 102 and through the opening 114 in
the emitter 110. To produce the electrospray, a sheath liquid can
be provided, flowing through the interior 116 of the emitter 110.
The sheath liquid can be delivered to the interior 116 from a
sheath liquid reservoir 120. A connecting fixture 124 can provide
the liquid communication between the sheath liquid reservoir 120
and the interior 116.
[0066] To provide the electrospray, the sheath liquid can flow
through the interior 116 of the emitter 110 past the distal end 106
of the capillary 102. When flowing past the distal end 106 of the
capillary 102, the sheath liquid interacts with the analyte
effluent expelled from the capillary 102 and forces the analyte
effluent toward the opening 114. The electrospray generated thus
includes a mixture of the analyte effluent and the sheath
liquid.
[0067] The electrospray can be generated by applying at least a
voltage HV2 between the sheath liquid reservoir 120 and the target
surface 130. The voltage HV2 drives electroosmotic flow of the
sheath liquid, using the zeta potential at the emitter 110 interior
surface. The HV2 voltage can provide electrokinetic flow sufficient
to generate an electrospray from the opening 114. The electrospray
can be a nanospray. The target surface 130 can be held at ground or
at a voltage.
[0068] In some embodiments, a voltage HV1 can be applied to the
capillary 102. The electrospray can then maintained by the electric
field at the emitter opening 114, which is generated by a
combination of voltage HV1 and HV2, or a surface potential at the
target surface 130. In such an arrangement, both HV1 and HV2
potentials contribute to the electric field at the emitter.
Ideally, the potential at the emitter is only controlled by HV2.
The resistance of the capillary is typically large enough that HV1
will have little effect on the electrospray. Instead,
electrophoresis is driven by the difference in potential between
HV1 and HV2. The HV2 voltage or the surface potential at the target
surface 130 are additional sources of potential that can be
regulated to maintain desired electric field at the emitter.
[0069] The capillary 102 can be used for capillary electrophoresis
(CE). When the interface is configured for CE, the voltage dropped
across HV1 to HV2 provides electrokinetic flow in the form of
electrokinetic separation for the analyte or analytes passing
through the capillary 102.
[0070] The sheath liquid can have electrical conductivity
properties such that an electrical connection sufficient to drive
the electrospray generating process is provided between the distal
end 106 of the capillary 102 and the distal end 112 of the emitter
110. The current generated by electrospray can be proportional to
the conductivity of the liquid. Representative sheath liquids
include, for example, 10 mM formic acid or acetic acid in 50%
methanol/acetonitrile or isopropanol. As would be recognized by
those of skill in the art, the percentage of organic solvents can
vary depending on the analytes of interest. Volatile salts, such as
ammonium formate or ammonium acetate, can be added to sheath
liquid.
[0071] The analyte can be a variety of composition of matter borne
by the analyte liquid through the capillary 102. The analyte may be
the analyte liquid itself, or the analyte may be dissolved within
the analyte liquid. Alternatively, the analyte may be
heterogeneously mixed with the analyte liquid. Representative
analytes include polar small molecules and salts thereof, and large
biomolecules (e.g., metabolites, peptides, proteins, lipids,
glycans, and nucleic acids). Other analytes include pesticides,
environmental contaminants, pharmaceuticals and their contaminants,
metabolites, and the like.
[0072] Further details of the individual components of the
interface 100 include the following.
[0073] The capillary 102 can be any suitable and effective
capillary for providing an analyte effluent. Representative
capillaries can be formed from glass (e.g., fused silica) or
plastic and can be cylindrical bodies having a tubular form wherein
the inner diameter of the capillary is on the order of about 0.5
microns to about 500 microns. In one embodiment, the inner diameter
is about 5 microns to about 75 microns, 5 microns to about 25
microns, 5 microns to about 15 microns, or about 10 microns.
[0074] The emitter 110 can be formed from glass, fused silica, and
any other suitable and effective material for emitters. Polymers,
such as TEFLON, can also be used, as can ceramics and any
non-conductive material that can be shaped to form the appropriate
structure of an emitter 110 as described herein. The emitter 110 is
typically a uniform cylinder prior to the taper towards the distal
end 112. However, the emitter 110 can also be tapered throughout,
or have a non-circular cross section.
[0075] The inner diameter of the emitter 110 must be larger than
the outer diameter of the capillary 102 so as to allow the
capillary to fit inside the emitter 110. The interior 116 of the
emitter 110 is defined by the space between the outer surface of
the capillary 102 and the inner surface of the emitter 110.
[0076] The opening 114 of the emitter 110 partially defines the
shape and size of the electrospray generated. In one embodiment,
the opening 114 in the distal end of the electrospray emitter is
about 0.5 microns to about 30 microns in diameter. The opening 114
is typically circular, although in certain embodiments the opening
is non-circular.
[0077] In one embodiment, the distal end of the capillary 106 and
the distal end of the electrospray emitter 112 (i.e., opening 114,
the electrospray emitter orifice) are separated by a distance of
less than about 750 microns. In another embodiment, the distal end
of the capillary 106 and the distal end of the electrospray emitter
112 are separated by a distance of less than about 700 microns,
less than about 500 microns, less than about 250 microns, less than
about 100 microns, less than about 50 microns, less than about 10
microns, less than about 5 microns, less than about 1 micron, or
less than about 0.5 microns. The distance between the distal end of
the capillary and the emitter can be as little as about 100 nm. In
one embodiment, the distance between the distal end of the
capillary and the emitter is about 150 microns to about 250
microns, or about 200 microns.
[0078] In another embodiment, the distal end of the capillary 106
can be configured to reside within the distal end of the
electrospray emitter 112. In addition, the distal end of the
capillary 106 can extend up to about 100 .mu.m beyond the emitter
orifice opening 114 provided that the outer diameter capillary 106
is smaller than the diameter of the emitter orifice opening 114. In
some embodiments, the distal end of the capillary 106 can extend
beyond the emitter orifice opening 114 by about 5 .mu.m, by about
25 .mu.m, by about 50 .mu.m, by about 75 .mu.m, or by about 100
.mu.m, or by a range between any two of the preceding values. This
configuration produces similar results as when the he distal end of
the capillary 106 and the distal end of the electrospray emitter
112 are separated by a distance of less than about 750 microns.
[0079] Valves, tubing, and other fluidic control components can be
used as the connecting fixture 124. An example of a representative
connecting fixture 124 is illustrated in FIG. 2. The connecting
fixture 124 can be made of TEFLON or other polymer tubing, glass,
or fused silica. A switching valve can be incorporated to switch
between multiple sheath fluids (i.e., between multiple sheath
liquid reservoirs 120), if desired. The dimensions of the
connecting fixture 124 are tied to the dimensions of the capillary
102 and the emitter 110. Representative inner diameters are about
10 .mu.m to about 5 mm, and a length of about 1 mm to about 30
cm.
[0080] Electrical potential typically drives the operation of the
interface 100. One embodiment provides the application of a
potential, HV2, at the sheath liquid reservoir 120, with a ground
or counter potential at the target surface 130. HV2 can be applied
to the sheath liquid reservoir 120 in a number of ways, such as by
electrical contact to an electrode disposed in the sheath liquid
reservoir 120. For example, a wire electrode can be submerged in
the sheath liquid reservoir 120 or an electrode can be disposed on
a wall of the sheath liquid reservoir 120.
[0081] In one embodiment, the analyte liquid is moved through the
capillary by a force such as an electrokinetic force or a
mechanical pumping force. Electrokinetic forces are well known to
those of skill in the art and include, but are not limited to,
dielectrophoresis, and electroosmotic or electrophoretic flow.
[0082] When electrokinetic flow is used, the optional voltage HV1
can be used. HV1 can be applied by making electrical contact to the
analyte liquid. For example, an electrode may be disposed at the
injection end 104 of the capillary 102, or a wire electrode can be
used to contact the analyte liquid near the injection end 104.
[0083] The analyte liquid can be separated within the capillary by
techniques such as capillary zone electrophoresis, capillary
electrochromatography, dielectrophoresis, or combinations thereof.
The analyte liquid can be separated by liquid chromatography prior
to entering the injection end of the capillary. The analyte liquid
need not always be separated when traveling through the capillary.
Instead, the capillary may receive pre-separated effluent and
simply transport the analyte in order to generate the electrospray.
Various chromatographic techniques can be used to provide the
analyte liquid to the capillary, as long as the effluent of the
chromatographic process can be provided and interfaced with the
injection end of the capillary.
[0084] In one embodiment, the analyte liquid is separated within
the capillary by electrophoresis, wherein the nanospray is produced
by electroosmotic flow, and wherein both the electrophoresis and
the electroosmotic flow are driven by applying an electric
potential between the injection end of the capillary, the sheath
liquid reservoir, and the target surface.
[0085] In some embodiments, the sheath-flow interface 100 is
configured to provide the nanospray to a mass spectrometer for
analysis (see FIG. 1B). In this embodiment, the target surface 130
is an input orifice of the mass spectrometer. Such a configuration
is described in greater detail in the examples below.
[0086] When the interface 100 is used for mass spectrometry, the
sheath liquid can be configured to enhance the compatibility of the
analyte effluent with the mass spectrometer. If analyte effluent is
not by itself compatible with MS, the sheath liquid can be selected
so as to facilitate effective MS. For example, if CE is used to
provide the analyte effluent, some common buffers may not be
compatible with MS. However, if a proper sheath liquid is used, the
buffer and analyte contained therein can be analyzed by MS.
[0087] The invention also provides methods for producing a
nanospray of an analyte effluent from a capillary using a
sheath-flow interface as described herein. In one embodiment the
method comprises applying a voltage to the sheath liquid reservoir
sufficient to drive electroosmotic flow of the sheath liquid from
the sheath liquid reservoir, through a connecting fixture
intermediate the capillary and the electrospray emitter, across the
distal end of the capillary, and through the opening at the distal
end of the electrospray emitter. The analyte effluent can be
separated within the capillary by capillary electrophoresis by
applying a voltage to the injection end of the capillary. However,
in other embodiments, the analyte effluent is separated by liquid
chromatography (or other separation method) prior to entering the
capillary. In yet other embodiments, the analyte effluent is not
separated either before entering or while traveling through the
capillary. As described above, the interface can be used as an
ionization source for mass spectrometry where the electrospray is
ionized and directed into an input orifice of a mass spectrometer
for analysis.
Design and Testing of CE-MS Nanospray Interface
[0088] This disclosure provides a nanospray sheath flow interface
in which a stable spray is achieved with very low sheath flow
rates, optionally without a pump or nebulizer gas. The separation
capillary can be placed inside an emitter, such as a tapered glass
ES emitter. Sheath liquid can be driven by electroosmosis produced
by the zeta potential at the emitter surface. The sheath liquid
flows over the end of the separation capillary, closing the circuit
and mixing with the capillary effluent inside the tip. The
capillary, the electrospray emitter, and sheath liquid tubing can
be connected, for example, by a PEEK cross. A small emitter tip
size (2 to 10 .mu.m i.d.) allows for operation in the nanospray
regime.
[0089] In previous interface designs, electrospray voltage is
applied directly to the electrospray tip, which often requires the
use of metal or metal-coated emitters or a wire electrode inside an
emitter. The lifetime of these metal-coated emitter is finite and
they often must be replaced. Although metal emitters are more
robust than glass, redox reactions on the metal surface often lead
to bubble formation and corona discharge, which limits the
electrospray sensitivity and stability. Wire electrodes also have
their limitations. For example, they can create turbulent flow and
loss of separation efficiency. Carbon-coated and conductive polymer
coated capillaries have been used to supply current to the tip.
However, carbon coatings have limited lifespans and must be
periodically replaced. Furthermore, conductive polymers must be
applied to the exterior of the emitter, which applications may lead
to emitter tip blockage. These problems can be circumvented by
applying voltage to the tip indirectly, for example, by a platinum
electrode placed in a sheath buffer reservoir.
[0090] Interface. A schematic of an interface is shown in FIG. 1B.
The separation capillary can be threaded into a glass electrospray
emitter using a cross fitting (e.g., 5-mm-long cross channels with
0.5 mm i.d.). One arm of the cross can be fitted with HPFA tubing
(0.5 mm i.d.) that has been dipped into a microcentrifuge tube,
which acts as the sheath liquid reservoir. The height of the liquid
in the reservoir can be kept at the same height as the emitter tip
to avoid hydrodynamic flow. High voltage can be applied to the
injection end of the capillary (HV1) and to the sheath liquid
reservoir (HV2). The separation can be driven by the difference in
these potentials. HV2 can provide the potential that drives
electro-osmotic flow in the emitter. The mass spectrometer inlet
can be held at ground potential. The other arm of the cross can be
used to flush the interface with sheath fluid at the start of an
experiment.
[0091] In various embodiments, electrospray emitters can be pulled
from 10 cm long borosilicate glass capillaries (1 mm o.d., 0.75 mm
i.d.), for example, with a P-1000 Sutter Puller. The tip sizes used
can range from about 2 .mu.m to about 50 .mu.m i.d., or about 2
.mu.m to about 10 .mu.m i.d., with a taper length of about 3 mm.
The tip diameter can be determined by inspection of the tip under a
microscope. The outer diameter of the tip can be noted, and the
inner diameter can be determined based on the ratio of the inner to
outer diameter of the initial glass capillary.
[0092] A detailed drawing of one example of an interface is shown
in FIG. 2. A fused silica separation capillary (50 .mu.m i.d., 150
.mu.m o.d., 45 cm long) can be threaded through a PEEK coned port
cross (Upchurch). The capillary is then threaded into the
electrospray emitter. The capillary and emitter tip can be held in
place with a sleeve, ferrule, and matching nut. The distance from
the capillary tip to the emitter tip can be adjusted while viewing
the system under a microscope as the fittings are tightened. Sheath
liquid is introduced into the electrospray tip by an HPFA tubing
connected to the cross. The fourth port of the cross can be
connected to a syringe and used to flush the interface with sheath
liquid at the start of an experiment.
[0093] Voltage can be delivered from a power supplies (e.g., a
Spellman CZE 1000R) by platinum electrodes that are in contact with
the running buffer and to the sheath liquid reservoir. The cross
can be attached to a translation stage placed at the source of a
Thermo LCQ ion trap instrument.
[0094] CE-MS. CE-MS experiments can be performed with instruments
such as a Thermo Finnigan LCQ ion trap instrument. Samples can be
biomolecules, such as an enzyme or peptide. Capillaries can be
conditioned with 0.1 M HCl, followed by 1 M NaOH, and then
separation buffer, all injected under 10 psi pressure for 5 minutes
before use.
[0095] In continuous infusion experiments, the sample can be
electrokinetically introduced into the separation capillary.
Continuous infusion can be used to tune the instrument for optimal
peptide signal. For capillary electrophoresis analysis, samples can
be electrokinetically injected. In various experiments, the emitter
tip can be placed about 2 mm away from the ion source. One set of
effective tuning parameters include a lens voltage of -38 V and
capillary temperature of 165.degree. C.
[0096] In positive ion mode experiments, the separation buffer can
be, for example, about 10 mM ammonium acetate, pH 7.8, and the
sheath liquid can be an equal volume mixture of about 10 mM aqueous
acetic acid and methanol.
[0097] In negative ion mode experiments, an Ultratrol LN (Target
Discovery, TM) coating can be applied to the capillary to minimize
electroosmotic flow. The separation buffer can consist of 10 mM
ammonium acetate, pH 8, and the sheath liquid can be an equal
volume mixture of 10 mM aqueous ammonium acetate and methanol.
[0098] With equal electroosmotic flow, the ratio of the electric
potential of separation buffer and sheath liquid can be
proportional to the ratio of the separation buffer and sheath
liquid flow rates.
[0099] Distance Between Capillary Exit and the Emitter Tip. The
distance from the capillary exit to the emitter exit significantly
affects the performance of the ESI emitter. U.S. Patent Publication
No. 2013/0140180 (Dovichi et al.) describes a sheath-flow interface
and experiments were performed where the distance between the
distal end of the separation capillary and the tip of the emitter
was reduced from 2 mm to 1 mm, which narrowed and increased peak
amplitude as a result of reduced extra-column band broadening.
However, when using the apparatus and techniques of U.S. Patent
Publication No. 2013/0140180 (Dovichi et al.), the inner diameter
of the tip of the emitter and the outer diameter of the capillary
resulted in a physical barrier that prevented the distal end of the
capillary from approaching closer than 800 .mu.m from the tip of
the emitter. This extremely close proximity of the outer diameter
edge of the distal end of the capillary and the inner diameter of
the tip of the emitter resulted in degraded system performance,
including decreased signal amplitude, broader peaks in
chromatographic performance, and decreased plate count.
[0100] Attempting to bring the distal end of the capillary closer
to the emitter tip is physically prevented by contact with the
emitter wall. This problem is particularly serious when using large
(e.g., 375 micron) outer diameter capillaries found in many
commercial electrophoresis instruments. Those capillaries require
separation distance between the capillary exit and the emitter
orifice of several millimeters, which dramatically decreases
sensitivity. Bringing the distal end of these large outer diameter
capillaries closer to the emitter tip can result in breakage of the
capillary tip, requiring disassembly and reassembly of the
instrument.
[0101] Solutions to this problem are described herein, whereby
reducing the outer diameter of the capillary, such as by etching
the outer diameter of a glass capillary, the distal end of the
capillary can be brought closer to the emitter orifice. By careful
etching of the outer surface of the capillary, the capillary can be
brought as close as 100 nm from the tip of the emitter (i.e., by
further etching of the embodiment illustrated in FIG. 3B). When the
distal end of the capillary is modified such that its outside
diameter is smaller than the diameter of the emitter orifice, the
tip of the emitter can also be configured to reside within the
emitter orifice and up to 100 microns beyond the emitter orifice.
This shorter spacing between the tip of the separation capillary
and the tip of the emitter, or its extension through the emitter
orifice, results in significantly less extra-column band
broadening.
[0102] Unexpectedly, the modification resulted in a two-order of
magnitude improvement in the state of art for bottom-up protein
identification and a one-order of magnitude improvement in the
state of art for MS-based peptide detection limits. The increased
sensitivity allows detection of peptides from very small amounts of
sample. In addition, given the same amount of material, this
interface will allow detection of more peptides and proteins than
the conventional design. Also unexpected was the cleaner background
from the mass spectrometer detection and the longer spray emitter
lifetime. The modifications to the interface result in an interface
such that very small sample sizes (e.g., 4-400 fg of peptides) can
be analyzed in less than 12 minutes of mass spectrometer instrument
time. Mass spectrometer detection limits as low as about 1
zeptomole (.about.600 molecules) can be obtained, thus masses as
low as approximately 1 attogram can be detected. Accordingly, the
enhanced sensitivity allows for the analysis of smaller samples, as
described herein, and to identify more peptides from the same size
sample used with previous interface designs.
[0103] The following Examples are intended to illustrate the above
invention and should not be construed as to narrow its scope. One
skilled in the art will readily recognize that the Examples suggest
many other ways in which the invention could be practiced. It
should be understood that numerous variations and modifications may
be made while remaining within the scope of the invention.
EXAMPLES
Example 1
Ultrasensitive and Fast Bottom-Up Analysis of Femtogram Amounts of
Complex Proteome Digests
[0104] Capillary zone electrophoresis was coupled with an improved
sheath-flow electrospray interface to produce a two order of
magnitude improvement in the amount of material required for
bottom-up protein analysis. This example describes an
ultrasensitive capillary zone electrophoresis-mass spectrometry
system based on the improved nanospray interface. The system can be
used for analysis of picogram to femtogram amounts of digests such
as E. coli digests. Over 100 proteins were identified based on
tandem mass spectra from 16 pg digests; over 60 proteins were
identified from 400 fg digests based on accurate mass and time tags
in 10 minutes.
[0105] Bottom-up proteomics of nanogram samples using capillary
liquid chromatography (LC)-electrospray ionization (ESI)-tandem
mass spectrometry (MS/MS) analyses requires at least one hour of
instrument time. In this example we report an ultrasensitive and
fast capillary zone electrophoresis (CZE)-ESI-MS/MS system based on
an improved electrokinetically-pumped sheath-flow interface. We
demonstrate the system for the rapid bottom-up analysis of
femtogram amounts of the E. coli protein digest.
[0106] CZE-ESI-MS/MS consistently outperforms LC-MS/MS for low
nanogram samples, in part because of its improved interface. This
example describes the development of another interface based on an
electrokinetically pumped sheath-flow interface (FIG. 3A) (see
Wojcik et al., Rapid Commun. Mass Spectrom. 2010, 24, 2554 for an
example of a previous sheath-flow interface). The new interface has
several advantages, including reduced sample dilution due to a very
low sheath flow rate, elimination of mechanical pumps, tolerance
for use of a wide range of separation buffers, and stable operation
in the nanospray regime. We have also coupled CZE to a
triple-quadrupole mass spectrometer with an interface for
quantification of Leu-enkephalin in a complex mixture using
multiple-reaction monitoring, and we obtained a 335 zmole peptide
detection limit, indicating this system's ability for high
sensitivity analysis (Li et al., Anal. Chem. 2012, 84, 6116).
[0107] A COMSOL model of the electrokinetically pumped sheath-flow
interface predicted and experiments verified that sensitivity
increases as the distal end of the capillary is brought closer to
the emitter orifice. A typical minimum distance between the
capillary tip and orifice is about 1 mm when using 150 micrometer
outer diameter capillary and 2.5 mm when using 375 micrometer outer
diameter capillary. The lower threshold of the distance is limited
by the outer diameter of the separation capillary, which eventually
butts against the interior of the conical emitter wall.
[0108] As described herein, we etched a few millimeters of the
outside of a separation capillary tip with hydrofluoric acid to
reduce its outer diameter from about 150 .mu.m to about 60 .mu.m or
about 50 .mu.m. This simple step allows us to place the capillary
end much closer to the emitter orifice (e.g., .about.200 .mu.m)
(FIGS. 3B and 3C), which results in a dramatic improvement in the
system's sensitivity. We used uncoated fused silica capillaries (32
cm and 40 cm, 10 .mu.m i.d./150 .mu.m o.d.) for electrophoresis,
and a Q-Exactive mass spectrometer for peptide identification.
Experimental details are provided in the Experimental section of
this example below.
[0109] Preparation of silica capillaries are described in the
Sutter Instrument Company Pipette Cookbook (2011, Novato, Calif.,
http://www.sutter.com/contact/faqs/pipette_cookbook.pdf). These
capillaries can then be etched to provide capillaries for the
interface described herein. An example of a useful capillary
according to one embodiment is a capillary with the following
dimensions: 25-30 cm long, 10 .mu.m i.d./150 .mu.m o.d., with an
etched end (.about.5 mm long, .about.50-60 .mu.m o.d.). For
capillaries with larger outer diameters (e.g., 250-300 .mu.m o.d.),
the outer diameter of the etched end can be reduced to about 200
.mu.m o.d., for example, where the capillary emitter is configured
to allow the distal end of the capillary to reside less than about
750 .mu.m from the emitter orifice. The distal ends of capillaries
can also be etched such that the outer diameter is reduce to about
20 .mu.m to about 100 .mu.m, about 20 .mu.m to about 80 .mu.m,
about 40 .mu.m to about 80 .mu.m, about 60 .mu.m to about 80 .mu.m,
about 40 .mu.m to about 60 .mu.m, or about 50 .mu.m to about 60
.mu.m.
[0110] We first evaluated the effect of separation voltage for the
analysis of 28 pg amounts of E. coli digests. Separations were
performed at 15 kV (500 V/cm) and 10 kV (300 V/cm) in a 32 cm-long
capillary. Electropherograms were generated with MaxQuant software
(v. 1.3.0.5) (Nat. Biotechnol. 26, 1367-1372 (2008)). The 10 kV
potential produced a wider separation window, which resulted in
significantly more protein (129.+-.18 vs. 88.+-.14) and peptide
(375.+-.27 vs. 246.+-.19) identifications compared with 15 kV. The
following work used an electric field of 300 V/cm.
[0111] We then evaluated the reproducibility of our CZE-ESI-MS/MS
system for analysis of 16 pg of the E. coli protein digests with a
40 cm capillary. We identified 105.+-.17 proteins and 256.+-.9
peptides based on triplicate bottom-up analysis of tandem mass
spectra. The state of the art for tandem mass spectra analysis of
complex protein digests is .about.100 protein identifications at
the 1 ng level. Our system produced a similar number of protein
identifications from two-orders of magnitude less sample.
[0112] The separations were reproducible and efficient. The signals
from 50 peptides were summed to produce extracted ion
electropherograms (FIG. 4). The average relative standard deviation
of the migration time of 154 peptides was 0.7% (FIG. 5) The
electrophoretic peaks were quite sharp, with an average width,
defined as the standard deviation of the Gaussian function used to
fit the peaks, of 0.7 s (1.6 s full width at half height) (FIG. 6).
We consistently obtained an average of over 300,000 theoretical
plates for the peptide separations (FIG. 7). Peak intensity was
also consistent between runs (FIG. 8). Separations were complete in
less than 10 minutes, which is an order of magnitude improvement in
analysis time compared to the state-of-the-art for high sensitivity
bottom-up proteomics of complex proteomes.
[0113] We next determined the relationship between the number of
identifications based on tandem mass spectra and the loaded amounts
of E. coli digests (FIG. 9). In duplicate 400 fg loadings, nine
peptides corresponding to 4.+-.1 proteins were confidently
identified after manual evaluation of tandem mass spectra. The most
abundant protein in E. coli, elongation factor Tu, makes up
.about.1% of the total protein mass. An analysis of 400 fg of an E.
coli digest will thus contain about 4 fg of this protein. The
minimum protein amount for identification by tandem mass
spectrometry is less than 4 fg, thus representing an improvement of
two orders of magnitude in the state of art (Shen et al., Anal.
Chem. 2004, 76, 144). When the sample loading amount was increased
to 84 pg, the number of protein and peptide identifications
increased to 162.+-.8 and 570.+-.11, respectively (FIG. 9).
[0114] We also applied Smith's accurate mass and time tag approach
using the 16 pg E. coli data as the database. Over 60 proteins and
150 peptides were identified from the 400 fg E. coli digests with
mass tolerance as 3 ppm, migration time tolerance as 0.3 min
(without alignment), and at least two detected isotopic peaks for
each peptide (FIG. 9). A representative extracted ion
electropherograms is shown in FIG. 10. This result is a 20-times
improvement in the number of protein identifications in the state
of art for AMTs based sub-picogram proteome analysis.
[0115] We finally estimated the peptide detection limit from the
400 fg E. coli data. We manually extracted electropherograms for
three peptides from elongation factor Tu, which were identified
based on MS/MS spectra with mass tolerance of 1 ppm. The
signal-to-noise ratios were obtained with Xcalibur software (Thermo
Fisher Scientific), using the noise region from 0.3 min to 2.3 min
after the peak (FIG. 11). Based on the amount of elongation factor
Tu present in the sample (4 fg) and its molecular weight (.about.43
kDa), .about.100 zmole of these peptides were taken for analysis.
These peptides generated signal-to-noise ratios (S/N) of
270.about.290; the mass detection limit (S/N=3) is .about.1 zmole
(.about.600 molecules), which is a one order of magnitude
improvement in the state of art for MS based peptide detection.
[0116] Explanations for the high sensitivity obtained from the
CZE-MS system include the following. First, the peptides only take
about 0.2 s or less to migrate from the capillary end to the spray
emitter end (approximately calculated based on our work), which
dramatically reduces the sample diffusion in the spray emitter and
generates a higher peptide signal, resulting in better peptide
detection limits. Second, the electroosmotic flow rate in the
separation capillary and in the spray emitter is approximately the
same due to similar buffer and applied voltage (.about.300 V/cm),
and the total flow rate for spray is around 20 nL/min, which
generates high ionization efficiency, resulting in high
sensitivity. Third, we employ quite narrow inner diameter
capillaries, which reduces sample flow rate and generates very
efficient separations.
[0117] We also note that the number of protein identifications
obtained in this work is .about.200 proteins due to the relatively
short peptide separation window, limiting the number of acquired
tandem spectra, which limits the detection of relatively low
abundance proteins in biological samples. Ways to improve the
protein identifications based on the CZE-MS system are to perform
online/offline peptide pre-fractionation before the CZE-MS
analysis, to use a longer separation capillary to slow the
separation, or to employ coated capillaries to reduce
electro-osmosis and increase the separation window.
[0118] In summary, this example describes an ultrasensitive and
high throughput CZE-ESI-MS/MS system for femtogram proteomics
analysis. The results obtained using the system are a one- to
two-orders of magnitude improvement in the amount of material
required for protein identification by tandem mass spectrometry, in
the number of proteins identified by accurate mass and time tags
from sub-picogram amounts of a complex protein digest, in peptide
mass detection limit, and in analysis time. The system can be used
for single cell analysis. To date, the highest sensitivity tools
available for single cell protein analysis have employed
laser-induced fluorescence detection. While sensitive, fluorescence
inherently generates a low-information content signal that provides
only rudimentary information on protein identity. The development
of CZE-mass spectrometry systems with 1-zmol detection limits opens
the door to single-cell protein analysis with confident
identification of relatively high abundance proteins.
[0119] Materials. Bovine pancreas TPCK-treated trypsin, urea,
ammonium bicarbonate (NH.sub.4HCO.sub.3), dithiothreitol (DTT), and
iodoacetamide (IAA) were purchased from Sigma-Aldrich (St. Louis,
Mo.). Acetonitrile (ACN), formic acid (FA), and hydrofluoric acid
(HF) were purchased from Fisher Scientific (Pittsburgh, USA).
Methanol and water were purchased from Honeywell Burdick &
Jackson (Wicklow, Ireland). Fused silica capillary (10 .mu.m
i.d./150 .mu.m o.d.) was purchased from Polymicro Technologies
(Phoenix, USA). Complete, mini protease inhibitor cocktail
(provided in EASYpacks) was purchased from Roche (Indianapolis,
USA). Equipment such as a PEEK cross, nuts, ferrules, sleeves and
PHFA tubing can be purchased from suppliers such as Idex Health and
Science (Oak Harbor, Wash., USA).
[0120] Preparation of the E. coli Sample. E. coli (Dh5-Alpha) was
cultured with the protocol described by Faserl et al. (Anal. Chem.
2011, 83, 7297). After culture, the E. coli pellets were first
washed with PBS (three times). Then, the pellets were suspended in
8 M urea and 100 mM Tris-HCl (pH 8.0) buffer containing protease
inhibitor and sonicated for 15 minutes on ice for cell lysis. The
lysate was centrifuged at 18,000 g for 15 minutes, followed by
protein concentration measurement with the BCA method (Cohen et
al., Annu. Rev. Anal. Chem. 2008, 1, 165). An aliquot of protein
(900 .mu.g) was precipitated by cold acetone overnight at
-20.degree. C. After centrifugation, the protein pellet was washed
again with cold acetone to remove contaminants. After incubation at
room temperature (.about.22.degree. C.) for several minutes to dry,
proteins were dissolved in 300 .mu.L of 8 M urea in 100 mM
NH.sub.4HCO.sub.3 (pH 8.5), followed by protein denaturation at
37.degree. C. for 60 minutes, reduction with DTT (8 mM) at
60.degree. C. for 1 hour, and alkylation with IAA (20 mM) at room
temperature for 30 minutes in the dark.
[0121] Then, 1.2 mL of 100 mM NH4HCO3 (pH 8.5) was added to dilute
the urea concentration to less than 2 M. Finally, an aliquot of 60
.mu.L treated protein solution (36 .mu.g) was digested overnight at
37.degree. C. with trypsin at a trypsin/protein ratio of 1/30
(w/w). The digests were acidified with formic acid (FA) (0.5% final
concentration) to terminate the reaction. The tryptic digests were
desalted with a ZipTipC18 (Millipore, Bedford, USA), followed by
lyophilization with a vacuum concentrator (Thermo Fisher
Scientific, Marietta, USA). The dried protein digests were
dissolved in 0.05% (v/v) FA aqueous buffer containing 20% (v/v)
ACN, resulting in solutions of 0.1 mg/mL and 0.01 mg/mL
concentration, which were then used for analysis.
[0122] Preparation of the separation capillary. A gentle flame was
used to remove a 1 cm length of the polyamide coating about 3-4 cm
from one end of a fused silica capillary (10 .mu.m i.d./150 .mu.m
o.d., .about.50 cm length). A reaction chamber was prepared by
drilling a small hole at the bottom of a 200 .mu.L Eppendorf tube
with a drill bit that is slightly larger than the capillary outer
diameter. The capillary was threaded through the hole so that the
flamed portion of the capillary was held within the Eppendorf
tube.
[0123] Finally, .about.150 .mu.L of hydrofluoric acid (HF,
.about.50% w/w) was added to the Eppendorf tube, and incubated at
room temperature in the hood for .about.20 minutes. After etching,
the outer diameter of the etched region was about 60 .mu.m, while
the inner diameter of the capillary was unchanged because it did
not come in contact with the HF. The exterior of the capillary was
washed with deionized water to remove the residual HF and then the
capillary was cut to .about.40 cm length with a .about.5 mm etched
region at the distal end. Caution and appropriate safety procedures
should be used while handling HF solutions. The etched capillary
was successively flushed with sodium hydroxide (1 M), deionized
water, hydrochloric acid (1 M), deionized water, and 0.5% (v/v)
FA.
[0124] CZE-ESI-MS/MS Analysis. The capillary electrophoresis system
was assembled from components reported previously (see Moini, Anal.
Chem. 2007, 79, 4241; Li et al., Anal. Chem. 84, 1617-1622 (2012);
Sun et al., Anal. Chem. 85, 4187-4194 (2013)). See also FIG. 3A.
Two Spellman CZE 1000R high-voltage power supplies provided high
voltage for the separation and electrospray. Voltage programming
was controlled by LabView software.
[0125] The total length of separation capillary was 40 cm (for 400
fg, 4 pg, and 16 pg amounts of E. coli digests) or 32 cm (for 28 pg
and 84 pg amounts of E. coli digests). The separation buffer was
0.5% (v/v) FA in deionized water, and the sheath liquid was 0.1%
(v/v) FA containing 10% (v/v) methanol. The sample was dissolved in
an aqueous buffer containing 0.05% (v/v) FA and 20% (v/v)
acetonitrile. The sample was injected into the separation capillary
by air pressure (10-20 psi and 1-9 s), and the sample injection
volume was calculated based on Poiseuille's law.
[0126] An electrokinetically pumped sheath flow interface was used
to couple the capillary to the mass spectrometer. The electrospray
emitter was drawn from a borosilicate glass capillary (1.0 mm o.d.,
0.75 mm i.d., and 10 cm length) pulled with a Sutter instrument
P-1000 flaming/brown micropipette puller. The emitter tip was
.about.8 .mu.m in outer diameter and 6 .mu.m inner diameter with a
3 mm taper. The etched end of the separation capillary was threaded
through a PEEK coned port cross (Upchurch, Oak Harbor USA) into the
spray emitter. The distance between the capillary tip and the
emitter end was about 200 .mu.m (see FIGS. 3B and C). Sheath liquid
was introduced into the electrospray tip by HPFA tubing connected
to the cross. The fourth port of the cross was connected to a
syringe and used to flush the emitter at the start of an
experiment.
[0127] The spray voltage (HVII, FIG. 3A) was 1.2 kV in each
experiments. High voltage I (HVI, FIG. 3A) was 11.2 kV for 32 cm
capillary (and 13.2 kV for 40 cm capillary) to produce an electric
field of .about.300 V/cm across the separation capillary. An
electric potential of 16.2 kV was also applied as HVI for 28 pg
amounts of E. coli digests analysis while optimizing the separation
conditions.
[0128] To obtain different sample loading amounts, 0.01 mg/mL E.
coli digests in 0.05% (v/v) FA aqueous buffer containing 20% (v/v)
ACN were used as the sample for 400 fg experiments, and 0.1 mg/mL
E. coli digests were used as the sample for 4- and 84-pg
experiments. Each sample was analyzed in duplicate or
triplicate.
[0129] A Q-Exactive mass spectrometer (Thermo Fisher Scientific)
was used in experiments in data dependent acquisition mode. Full MS
scans were acquired in the Orbitrap mass analyzer over m/z
380-1,800 (for 28 and 84 pg E. coli samples) and m/z 400-1,000 (for
400 fg, 4 pg, and 16 pg of E. coli samples) with resolution 70,000
(at m/z 200). The target values and maximum injection time were
1.00E+06 and 250 ms (for 16, 28, and 84 pg of E. coli samples);
3.00E+06 and 500 ms (for 400 fg and 4 pg E. coli samples).
[0130] For 400 fg of E. coli digests analysis, two most intense
peaks (Top 2 method) with charge state as 2 or 3 and intensity
higher than 2.00E+03 were sequentially isolated in the quadrupole
with isolation window as 2 m/z and fragmented in the higher energy
collisional dissociation collision cell with normalized collision
energy of 28%, and tandem mass spectra were acquired in the
Orbitrap mass analyzer with resolution 35,000 (m/z 200), target
value 1.00E+06, and maximum injection time 500 ms.
[0131] For 4 pg of E. coli digests analysis, the Top 4 method was
applied, and maximum injection time for tandem spectra as 250 ms.
For 16 pg of E. coli digests analysis, the Top 6 method was used.
The maximum injection time for tandem spectra was 120 ms, intensity
threshold was 4.00E+03, and charge exclusion was 1 and 5 and
higher. For 28 and 84 pg of E. coli digests analysis, the Top 6
method was used. The maximum injection time for tandem spectra was
120 ms, intensity threshold was 8.00E+03, and charge exclusion was
1 and 7 and higher. For each experiment, dynamic exclusion was 6.0
s, microscans was 1, peptide match was on, and exclude isotopes was
on.
[0132] Data Analysis. Raw MS files were analyzed by MaxQuant
software version 1.3.0.5. MS/MS spectra were searched by the
Andromeda search engine (Cox et al., J. Proteome Res. 10, 1794)
against the NCBI-E. coli (DH1) database containing forward and
reverse sequences (8,320 entries including forward and reverse
sequences). The database also included common contaminants.
MaxQuant analysis included an initial search with a precursor mass
tolerance of 10 ppm, main search precursor mass tolerance of 5 ppm,
and fragment mass tolerance of 20 ppm. The search included the
enzyme as trypsin, variable modifications of methionine oxidation,
N-terminal acetylation and deamidation (NQ), and fixed modification
of carbamidomethyl cysteine. Minimum peptide length was set to
seven amino acids and the maximum number of missed cleavages was
set to two. The false discovery rate was set to 0.01 for both
peptide and protein identifications. The proteins identified by the
same sets of peptides were reported as one protein group. The
protein and peptide tables were filtered to remove the
identifications from the reverse database and common
contaminants.
Example 2
CZE-ESI-MS/MS for Quantitative Parallel Reaction Monitoring
[0133] This example describes the use of capillary zone
electrophoresis-electrospray ionization-tandem mass spectrometry
(CZE-ESI-MS/MS) for the quantitative parallel reaction monitoring
of peptide abundance and single-shot proteomic analysis of a human
cell line.
[0134] We coupled capillary zone electrophoresis (CZE) with an
ultrasensitive electrokinetically pumped nanospray ionization
source for tandem mass spectrometry (MS/MS) analysis of complex
proteomes. We first used the system for the parallel reaction
monitoring (PRM) analysis of angiotensin II spiked in 0.45 mg/mL of
bovine serum albumin (BSA) digest. A calibration curve was
generated between the loading amount of angiotensin II and
intensity of angiotensin II fragment ions. CZE-PRM generated a
linear calibration curve across over 4.5 orders of magnitude
dynamic range corresponding to angiotensin II loading amount from 2
amole to 150 fmole. The relative standard deviations (RSDs) of
migration time were <4% and the RSDs of fragment ion intensity
were .about.20% or less except 150 fmole angiotensin II loading
amount data (.about.36% RSD).
[0135] We further applied the system for the first bottom up
proteomic analysis of a human cell line using CZE-MS/MS. We
generated 283 protein identifications from a 1 hour long,
single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line
digest, corresponding to an approximately 80 ng loading amount. The
MCF7 digest was fractionated using a C18 solid phase extraction
column. Single-shot analysis of a single fraction resulted in 468
protein identifications, which is by far the largest number of
protein identifications reported for a mammalian proteomic sample
using CZE.
[0136] Capillary electrophoresis (CE)-electrospray ionization
(ESI)-mass spectrometry (MS) has been used to characterize a wide
range of analytes, including intact proteins, peptides, and
metabolites. One useful CE-nanospray interface is a sheath-flow
interface we developed that employs a glass emitter with a
.about.5-.mu.m orifice (Wojcik et al., Rapid Commun. Mass Spectrom.
24 (2010) 2554). Electro-osmosis at the glass surface drives the
sheath fluid at very low rates. The interface has several
advantages, including minimal sample dilution due to the very low
sheath flow rate, elimination of mechanical pumps and nebulizing
gas, use of a wide range of separation buffers, and stable
operation in the nanospray regime. We have applied the
electrokinetically pumped sheath flow nanospray interface CE-MS/MS
system for shot-gun proteomic analysis of the secretome of
Mycobacterium marinum (Li et al., Anal. Chem. 84 (2012) 1617), a
fraction of yeast lysate (Wojcik et al., Talanta 88 (2012) 324),
the E. coli proteome (Zhu et al., Anal. Chim. Acta. 810 (2014) 94),
picogram amounts of RAW 264.7 cell lysate (Sun et al., Analyst 138
(2013) 3181), and the PC12 cell lysate (Zhu et al., Anal. Chem. 85
(2013) 7221). In addition, the system was also applied for top-down
intact protein characterization (Sun et al., Anal. Chem. 85 (2013)
5989), quantitative multiple reaction monitoring (MRM) of peptide
abundance (Li et al., Anal. Chem. 84 (2012) 6116), and
phosphopeptides characterization (Mou et al., Anal. Chem. 85 (2013)
10692).
[0137] In Example 1 above, we described a simple modification to
our interface that results in ultrasensitive performance. We etched
a few millimeters of the outside of the separation capillary tip
with hydrofluoric acid to reduce its outer diameter from .about.150
.mu.m to .about.60 .mu.m. This step allows the capillary tip to be
placed within 200 .mu.m of the emitter orifice, which results in a
significant improvement in the system's sensitivity (Sun et al.,
Angew. Chem. Int. Ed. Engl. 52 (2013) 13661). By employing a 10
.mu.m i.d. separation capillary, a Q-Exactive mass spectrometer,
and the improved CE-MS interface, we obtained 1 zmole (1
zmol=10.sup.-21 mol=.about.600 molecule) peptide detection limit
(S/N=3). Over 100 proteins were identified based on tandem mass
spectra from 16 pg of E. coli digest and 154 peptides from 60
proteins were identified from 400 fg sample loading.
[0138] Our group used the electrokinetically pumped sheath flow
interface for the capillary zone electrophoresis analysis of the
secretome of M. marinum in 2012; 140 protein groups were identified
(Li et al., Anal. Chem. 84 (2012) 1617). We improved the peptide
separation by using linear polyacrylamide coated capillary and
stacking injection. Approximately 300 protein groups were
identified from 100 ng of E. coli digests by single shot analysis
in less than 1 h (Zhu et al., Anal. Chem. 85 (2013) 2569). The
number of protein IDs was increased to 871 by analyzing seven E.
coli digest fractions from offline C18-SPE fractionation (Yan et
al., Proteomics 13 (2013) 2546). We also employed a capillary
isoelectric focusing MS/MS system with the electrokinetically
pumped sheath flow interface for eight-plex iTRAQ based
quantitative proteomic analysis of differentiating PC12 cells; 835
protein groups were identified (Zhu et al., Anal. Chem. 85 (2013)
7221). To our knowledge, there are no publications employing
CZE-MS/MS for analysis of a human cell line.
[0139] For target proteomics research, multiple/selected reaction
monitoring (MRM/SRM) is typically employed with triple-quadrupole
(QqQ) mass spectrometer. Briefly, the parent ion of a targeted
peptide is isolated in the first quadrupole (Q1) and then
fragmented in the second quadrupole (Q2). One or several fragment
ions from the targeted peptide are further isolated by the third
quadrupole (Q3) for detection. Recently, a new target proteomics
technique was introduced, named parallel reaction monitoring (PRM),
which was performed with a bench-top quadrupole-Orbitrap mass
spectrometer (Peterson et al., Mol. Cell. Proteomics 11 (2012)
1475). For PRM, a target peptide is selected in the quadrupole and
then fragmented in the collisional cell. The resulting fragment
ions are analyzed in the Orbitrap to generate one full,
high-resolution MS/MS spectrum. Because m/z ratios of fragment ions
are not required during the method development step, the process is
much easier than SRM/MRM. In addition, PRM has much better
tolerance to the background matrix than SRM/MRM due to the high
resolution of the Orbitrap analyzer.
[0140] In this example, we describe the first example of CZE-PRM.
We employ our improved electrokinetically pumped sheath flow
nanospray interface described above in Example 1 for peptide
analysis. A standard peptide, angiotensin II, was spiked in a 0.45
mg/mL bovine serum albumin digest to evaluate the CZE-PRM system
performance. We observed over four and a half orders of magnitude
linear dynamic range for angiotensin II corresponding to loading
amounts from 2 to 150,000 amole. We also presented the first
example of CZE-MS/MS for bottom-up analysis of a human cell line.
Nearly 300 proteins were identified from MCF7 whole cell lysate
digest in a 1-hour single-shot CZE-MS/MS analysis with .about.80 ng
loading amount.
[0141] Experimental
[0142] 1. Materials and reagents. Bovine pancreas TPCK-treated
trypsin, bovine serum albumin (BSA), urea, ammonium bicarbonate
(NH.sub.4HCO.sub.3), dithiothreitol (DTT), iodoacetamide (IAA), and
angiotensin II (human, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) were
purchased from Sigma-Aldrich (St. Louis, Mo.). Acetonitrile (ACN),
formic acid (FA), and hydrofluoric acid (HF) were purchased from
Fisher Scientific (Pittsburgh, Pa.). Methanol and water were
purchased from Honeywell Burdick & Jackson (Wicklow, Ireland).
Fused silica capillary (10 and 20 .mu.m i.d./150 .mu.m o.d.) and
linear polyacrylamide (LPA) coated capillary (50 .mu.m i.d./150
.mu.m o.d.) were purchased from Polymicro Technologies (Phoenix,
Ariz.).
[0143] Eagle's minimal essential medium (EMEM), fetal bovine serum
(FBS), GlutaMAX.TM. (100.times.), insulin, and
Antibiotic-Antimycotic (Anti-Anti, 100.times.) were purchased from
Life Technologies Corporation (Grand Island, N.Y.). Mammalian
Cell-PE LB.TM. buffer for cell lysis was purchased from
G-Biosciences (St. Louis, Mo.). Complete, mini protease inhibitor
cocktail (provided in EASYpacks) was purchased from Roche
(Indianapolis, Ind.).
[0144] 2. Sample preparation. Bovine serum albumin (BSA, 0.5 mg/mL)
in 100 mM NH.sub.4HCO.sub.3 (pH 8.0) containing 8 M urea was
denatured at 37.degree. C. for 30 minutes, followed by standard
reduction and alkylation with DTT and IAA. After dilution with 100
mM NH.sub.4HCO.sub.3 (pH 8.0) to reduce the urea concentration
below 2 M, protein digestion was performed for 12 hours at
37.degree. C. with trypsin at a trypsin/protein ratio of 1/30
(w/w). After acidification, the protein digest was desalted with a
C18-SepPak column (Waters, Milford, Mass.), followed by
lyophilization with a vacuum concentrator (Thermo Fisher
Scientific, Marietta, Ohio). The dried sample was dissolved in
0.05% (v/v) FA to produce a 0.5 mg/mL solution and stored at
.about.20.degree. C. before use.
[0145] Angiotensin II solution was spiked into the BSA digest to
generate five samples in 0.05% (v/v) FA containing 0.45 mg/mL BSA
digest and different concentrations of angiotensin II (10 nM, 100
nM, 1 .mu.M, 10 .mu.M and 100 .mu.M). Each sample was analyzed by
CZE-PRM in triplicate.
[0146] The procedures for MCF7 cell culture, cell lysis, protein
acetone precipitation, denaturation, reduction and alkylation used
are described by Sun et al. (J. Chromatogr. A. 1337 (2014) 40).
Briefly, after cell culture, MCF7 cells were lysed by sonication,
followed by BCA protein concentration measurement, and acetone
precipitation. Then, a 690 .mu.g protein aliquot was dissolved in 8
M urea and 100 mM NH.sub.4HCO.sub.3 (pH .about.8.0), denatured at
37.degree. C. for 1 hour, and reduced in .about.40 mM DTT at
37.degree. C. for 1.5 hours, followed by alkylation with 100 mM IAA
at room temperature for 30 minutes. After dilution with 100 mM
NH.sub.4HCO.sub.3 (pH .about.8.0) to reduce the urea concentration
below 2 M, the proteins were digested by trypsin at a
trypsin/protein ratio of 1/30 (w/w) overnight at 37.degree. C. The
protein digest was acidified with FA (1% final concentration), and
desalted with C18-SepPak column (Waters, Milford, Mass.), followed
by lyophilization. The peptide mixture was dissolved in 0.04% (v/v)
FA containing 20% (v/v) ACN to get a 3.5 mg/mL sample, followed by
CZE-ESI-MS and MS/MS analysis. Around 300 .mu.g of digest was
lyophilized again, and redissolved in 0.1% (v/v) FA. The digest was
loaded onto a C18-SepPak SPE column (Waters), and eluted by 1 mL of
12% (v/v) ACN containing 0.1% (v/v) FA. The eluate was lyophilized
and further dissolved in 28 .mu.L of 0.04% (v/v) FA and 30% (v/v)
ACN for CZE-ESI-MS/MS analysis.
[0147] 3. CZE-ESI-MS/MS analysis. The capillary electrophoresis
system was assembled from components above in Example 1. Two
Spellman CZE 1000R high-voltage power supplies provided voltages
for separation and electrospray. An ultrasensitive
electrokinetically pumped sheath flow interface was used to couple
CZE to a Q-Exactive mass spectrometer (Thermo Fisher Scientific).
The electrospray emitter was a borosilicate glass capillary (1.0 mm
o.d., 0.75 mm i.d.) pulled with a Sutter instrument P-1000
flaming/brown micropipette puller, and the size of the emitter
orifice was 8-10 .mu.m. Voltage programming was controlled by
LabView software.
[0148] For the CZE-PRM experiment, a 32 cm long capillary (10 and
20 .mu.m i.d./150 .mu.m o.d.) with etched tip (.about.60 .mu.m
o.d., .about.5 mm length) was used for the separation. The
separation buffer was 0.5% (v/v) FA. The voltage at the injection
end was 16.2 kV, and 1.2 kV was applied as spray voltage. For whole
MCF7 cell lysate digest analysis, a 100 cm long capillary (20 .mu.m
i.d./150 .mu.m o.d.) with .about.5 mm length etched end (.about.60
.mu.m o.d.) was employed, and the separation buffer was 0.5%(v/v)
FA. For the MS1 only experiment, 29.2 kV was applied at the
injection end; for MS/MS experiment in data dependent acquisition
(DDA) mode, 21.2 kV was applied at the injection end. In both
cases, 1.2 kV was applied for electrospray.
[0149] For analysis of the 12%(v/v) ACN eluate of MCF7 cell lysate
digest from C18-SPE column, an LPA-coated capillary (62 cm, 50
.mu.m i.d./150 .mu.m o.d.) with .about.5 mm long etched end
(.about.70 .mu.m o.d.) was used, and the separation buffer was
0.12% (v/v) FA. The voltage applied at injection end was 16.2 kV
and 1.2 kV was applied as spray voltage.
[0150] For all the experiments, the sheath buffer was 10% (v/v)
methanol and 0.1% (v/v) FA. The distance between the separation
capillary end and spray emitter tip was around 200 .mu.m. The
sample was injected into the separation capillary by nitrogen
pressure, and the injection volume was calculated based on
Poiseuille's law. All the HF etching operations for separation
capillaries were performed with the same protocol as described
above in Example 1. The etched 10 and 20 .mu.m i.d. separation
capillary was successively flushed with sodium hydroxide (1 M),
deionized water, hydrochloric acid (1 M), deionized water, and 0.5%
(v/v) FA before use. The etched LPA-coated separation capillary (50
.mu.m i.d.) was successively flushed with deionized water and 0.12%
(v/v) FA before use.
[0151] For CZE-PRM experiment, the Q-Exactive was programmed in
target-MS.sup.2 mode with inclusion list as on. In the inclusion
list, 523.7734 and +2 were set as the target m/z and charge,
respectively. The normalized collisional energy (NCE) was 25%. The
other parameters were as follows: resolution for MS/MS as 17,500,
AGC target as 5E5, a maximum injection time as 500 ms, isolation
window as 1 m/z, and microscans as 1.
[0152] For analysis of the human cell line digest, both MS1-only
and regular DDA mode experiments were performed. For the MS1-only
experiment, MS spectra were acquired with 380-1800 m/z range,
70,000 resolution (at m/z 200), AGC target as 1E6, maximum
injection time as 250 ms, and microscans as 1. For the DDA mode
experiment, a top 12 method was employed. For MS1 full scan, the
parameters were the same as the MS1-only experiment. For MS/MS,
twelve most intense peaks from the MS spectrum were sequentially
isolated in the quadrupole (isolation window as 2.0 m/z) and
further fragmented in the higher energy collisional dissociation
(HCD) cell (NCE as 28%), followed by Orbitrap analysis. Resolution
of 35,000 (at m/z 200), AGC target as 1E6, maximum injection time
as 120 ms, and microscans as 1 were applied. The parent ions with
charge states higher than +1 and intensity higher than 8.3E3 were
chosen for fragmentation. Dynamic exclusion was set as 10 s for 20
.mu.m i.d. capillary and 15 s for 50 .mu.m i.d. capillary. Peptide
match and exclude isotopes were turned on.
[0153] 4. Data analysis. For CZE-PRM experiment, the data were
manually analyzed with Thermo Xcalibur software. The peaks of
fragment ions from angiotensin II were manually extracted with 10
ppm mass tolerance and Gaussian smoothing (5 points) was performed
on the peaks.
[0154] For human cell lysate digest data, the raw files containing
tandem spectra were analyzed with Proteome Discoverer 1.3 software
(Thermo Fisher Scientific). Mascot 2.2 was used for database
searching against Swiss-Prot database with taxonomy as human
(20,335 sequences). Trypsin was chosen as the digestion enzyme, and
the maximum number of missed cleavages was set as 2. The mass
tolerances for parent ions and fragment ions were set as 10 ppm and
0.05 Da, respectively. Dynamic modifications included oxidation
(M), deamidation (NQ), and acetylation (K and protein N-terminus).
Carbamidomethylation (C) was set as the fixed modification.
Database searching against the corresponding reversed database was
also performed in order to evaluate the false discovery rate (FDR)
(Elias and Gygi, Nat. Methods 4 (2007) 207).
[0155] Percolator software (version 1.17) integrated in the
Proteome Discoverer 1.3 was used to evaluate the database searching
results. For peptide level analysis, peptide confidence value as
high was used to filter the peptide identification, corresponding
to peptide-level FDR less than 1%. For peptides per protein
settings, the following parameters were applied, including minimal
number of peptides as 1, count only rank 1 peptides, and count
peptide only in top scored proteins. In addition, protein grouping
was enabled.
[0156] Results and Discussion
[0157] 1. CZE-PRM for peptide detection. For CZE-PRM experiments,
the Q-Exactive was programmed in target-MS.sup.2 mode. In the
inclusion list, m/z 523.7734 (+2) corresponding to angiotensin II
(human, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) was used. The angiotensin
II standard peptide was spiked into a 0.45 mg/mL BSA digest to
generate a series of samples containing 10 nM, 100 nM, 1 .mu.M, 10
.mu.M, and 100 .mu.M of angiotensin II, corresponding to 15 amole
to 150 fmole of angiotensin II loaded onto the separation capillary
(20 .mu.am i.d.) in the presence of a constant background of BSA
digest.
[0158] We also employed a 10 .mu.m i.d. separation capillary to
analyze a sample made from 10 nM angiotensin II spiked in a 0.45
mg/mL BSA digest. In this experiment, about 2 amole of angiotensin
II was loaded onto the capillary. After triplicate CZE-PRM
analyses, the two most intense fragment ions (b6.sup.+ and
y2.sup.+) of angiotensin II were extracted from the acquired data
with 10 ppm mass tolerance to construct a calibration curve (FIG.
12). We performed an unweighted weighting linear fit to the log-log
data. The fragment ion signal increased linearly with angiotensin
loading amount over 4.5 orders of magnitude (log-log b6.sup.+
slope=0.90, r=0.99; y2.sup.+ slope=0.89, r=0.99). We note that the
two fragment ions of angiotensin II co-migrate in each CZE-PRM run,
which confirms the detection of the standard peptide.
[0159] Use of 2 amole of angiotensin II in the presence of the 0.45
mg/mL (.about.7 .mu.M) BSA digest background generated
.about.2.0E+03 signal for the most abundant fragment ion (y2.sup.+)
of angiotensin II. This signal is .about.20 times higher than that
from our previous work that also injected .about.2 amole of
Leu-enkephalin, in the presence of 66 nM of BSA digest as
background and using CZE-MRM analysis (Li et al., Anal. Chem. 84
(2012) 6116).
[0160] The dramatic improvement in signal amplitude is caused by
several factors. First, our first-generation electrokinetically
pumped sheath flow interface was employed in the earlier work,
whereas the improved version of the interface (Example 1 above) was
used in this work. Because the distance between separation
capillary tip and spray emitter tip in the improved interface is
five-times shorter than that in original version, diffusion of
peptides in the spray emitter is decreased, peptide peaks are much
sharper, and peptide signals are correspondingly more intense.
Second, we used a separation capillary with much smaller inner
diameter (10-20 .mu.m vs. 50 .mu.m) and separation buffer with much
lower pH value (lower than 3 vs. 6-8), which reduces the flow rate
in the separation capillary, generating better sensitivity. Third,
online stacking was used in this work, which again sharpens peaks.
The conductivity of sample matrix (0.05% (v/v) FA) is much lower
than the separation buffer (0.5% FA), so that peptides are
concentrated when high voltage is applied across the capillary. In
addition, a much higher resolution mass analyzer (Orbitrap vs.
quadrupole) was employed in this work, which generates vastly
superior tolerance to the background matrix.
[0161] We also evaluated the reproducibility of the CZE-PRM system
in terms of migration time and intensity of angiotensin II fragment
ion (y2.sup.+) (FIG. 13). The relative standard deviations (RSDs)
of migration time for triplicate analysis were less than 4%. The
RSDs of the fragment ion intensity for triplicate analysis were
.about.36% for 150 fmole loading amounts, around 10% or less for
150 amole to 15 fmole loading amounts, and close to 20% for 2 and
15 amole loading amounts.
[0162] The slightly higher RSDs for the 2 and 15 amole loading
amount data are likely due to two reasons. First, the intensity of
co-isolated BSA peptides will be much higher than angiotensin II,
leading to higher detection variation for very low loading amounts.
Second, the very sharp peaks of the fragment ions (full width at
half height for the peaks are .ltoreq.2 s) for the 2 and 15 amole
loading amount data leads to fewer data points across the peaks
than the higher loading amounts data. For the 150-fmole loading
amount data, the RSD is significant higher because the CE-MS system
is slightly overloaded, which produces non-Gaussian peak shapes. In
addition, the RSDs data of migration time and fragment ion
intensity for angiotensin II fragment ion b6.sup.+ agree well with
that for y2.sup.+.
[0163] 2. CZE-ESI-MS and MS/MS for human cell lysate digest
analysis. Example 1 above described the use of an etched-tip
electrokinetically pumped sheath flow nanospray interface based
CZE-MS/MS system, which was used to analyze high-femtogram to
mid-picogram amounts of E. coli cell lysate digest. That study
demonstrated that the improved system was highly efficient and
quantitatively reproducible for separation and detection of a
prokaryote proteome digest. To date, there has been no published
bottom-up analysis of a human proteome using CZE-MS/MS.
[0164] Here, we report the first application of CZE-MS/MS for the
bottom-up analysis of a human proteome, the proteome of the MCF-7
human breast cancer cell line. We began by using the ultrasensitive
interface for analysis of .about.60 ng of a whole-cell digest (FIG.
14). The separation was driven by a 280 V/cm electric field.
Peptides began to migrate from the capillary at 11 minutes, and the
separation was nearly complete by 26 minutes (FIG. 14A), although a
few strong peaks were observed at later times. The separation is
highly efficient and peaks are very sharp (FIG. 14B).
[0165] Roughly 10-20 peaks were resolved from the base peak
electropherogram across the 1-minute separation window from 13-14
minutes. We extracted one peptide (m/z 400.77161, z=+2) from the
data with 2 ppm mass tolerance that generated 525,000 theoretical
plates, which demonstrates the extraordinarily high separation
efficient produced by this instrument.
[0166] We next applied single-shot CZE-MS/MS for bottom-up analysis
of the MCF7 whole cell lysate digest. Around 80 ng of peptides was
loaded onto a 100-cm long, 20 .mu.m i.d. separation capillary. The
separation was performed at 200 V/cm electric field. After database
searching of the tandem spectra, 1,159 peptides and 283 proteins
were confidently identified, and 176 proteins were identified based
on at least two peptides. The separation was complete in roughly 40
minutes.
[0167] To test how many peptides and proteins can be identified
from the MCF7 proteome with single-shot CZE-MS/MS, we simplified
the MCF7 cell lysate digest by fractionating the proteome. Peptides
were first trapped on a C18 SPE column and then a fraction was
eluted with 12% (v/v) ACN for analysis. An LPA-coated separation
capillary was used to reduce electro-osmosis, which increases the
separation capacity. To increase sample injection volume, we used a
50-.mu.m ID capillary and injected about 130 nL of sample. After
database searching, the single-shot analysis identified 1,199
peptides and 468 proteins. 219 proteins were identified based on at
least two peptides. The separation was complete within 60 minutes.
The combined datasets contain 2,005 peptide and 537 protein
IDs.
[0168] While specific embodiments have been described above with
reference to the disclosed embodiments and examples, such
embodiments are only illustrative and do not limit the scope of the
invention. Changes and modifications can be made in accordance with
ordinary skill in the art without departing from the invention in
its broader aspects as defined in the following claims.
[0169] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. No limitations inconsistent with this
disclosure are to be understood therefrom. The invention has been
described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that
many variations and modifications may be made while remaining
within the spirit and scope of the invention.
* * * * *
References